Metabolic Retroconversion of Trimethylamine N-Oxide and the Gut Microbiota

bioRxiv preprint doi: https://doi.org/10.1101/225581; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota 2 3 Lesley Hoyles1*, Maria L. Jiménez-Pranteda2*, Julien Chilloux1*, Francois Brial3, Antonis 4 Myridakis1, Thomas Aranias3, Christophe Magnan4, Glenn R. Gibson2, Jeremy D. Sanderson5, Jeremy 5 K. Nicholson1, Dominique Gauguier1,3, Anne L. McCartney2† and Marc-Emmanuel Dumas1† 6 7 1Integrative Systems Medicine and Digestive Disease, Department of Surgery and Cancer, Faculty of 8 Medicine, Imperial College London, Exhibition Road, London SW7 2AZ, UK 9 2Food Microbial Sciences Unit, Department of Food and Nutritional Sciences, School of Chemistry, 10 Food and Pharmacy, Faculty of Life Sciences, The University of Reading, Whiteknights Campus, 11 Reading RG6 6UR, UK 12 3Sorbonne Universities, University Pierre & Marie Curie, University Paris Descartes, Sorbonne Paris 13 Cité, INSERM UMR_S 1138, Cordeliers Research Centre, Paris, France 14 4Sorbonne Paris Cité, Université Denis Diderot, Unité de Biologie Fonctionnelle et Adaptative, CNRS 15 UMR 8251, 75205 Paris, France 16 5Department of Gastroenterology, Guy's and St Thomas' NHS Foundation Trust and King's College 17 London, London, UK 18 19 *These authors made equal contributions to this work; shared first authorship. 20 †Corresponding authors: Anne L. McCartney; Marc-Emmanuel Dumas 21 Keywords: co-metabolic axis; gut–liver axis; metabolomics; Enterobacteriaceae; lactic acid bacteria. 22 23 Email addresses: L. Hoyles, [email protected]; M.L. Jiménez-Pranteda, [email protected]; J. 24 Chilloux, [email protected]; F. Brial, [email protected]; A. Myridakis, 25 [email protected]; T. Aranias, [email protected]; C. Magnan, christophe.magnan@univ-paris- 26 diderot.fr; G.R. Gibson, [email protected]; J.D. Sanderson, [email protected]; J.K. 27 Nicholson, [email protected]; D. Gauguier, [email protected]; A.L. McCartney, 28 [email protected]; M.-E. Dumas, [email protected] 29 1 bioRxiv preprint doi: https://doi.org/10.1101/225581; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 30 ABSTRACT 31 BACKGROUND: The dietary methylamines choline, carnitine and phosphatidylcholine are used by 32 the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However, 33 little is known about the use of trimethylamine N-oxide (TMAO) by this consortium of microbes. 34 RESULTS: A feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial 35 conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to 36 TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated 37 mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture 38 fermentation systems inoculated with human faeces, growth of Enterobacteriaceae was stimulated in 39 the presence of TMAO. Human-derived faecal and caecal bacteria (n = 66 isolates) were screened on 40 solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by 1H- 41 NMR. As with the in vitro fermentation experiments, TMAO stimulated the growth of 42 Enterobacteriaceae; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates 43 of Escherichia coli produced more TMA from TMAO than their faecal counterparts. Lactic acid 44 bacteria produced increased amounts of lactate when grown in the presence of TMAO, but did not 45 produce large amounts of TMA. Clostridia (sensu stricto), bifidobacteria and coriobacteria were 46 significantly correlated with TMA production in the mixed fermentation system but did not produce 47 notable quantities of TMA from TMAO in pure culture. 48 CONCLUSIONS: Reduction of TMAO by the gut microbiota (predominantly Enterobacteriaceae) 49 to TMA followed by host uptake of TMA into the bloodstream from the intestine and its conversion 50 back to TMAO by host hepatic enzymes is an example of metabolic retroconversion. TMAO 51 influences microbial metabolism depending on isolation source and taxon of gut bacterium. 52 Correlation of metabolomic and abundance data from mixed microbiota fermentation systems did not 53 give a true picture of which members of the gut microbiota were responsible for converting TMAO to 54 TMA; only by supplementing the study with pure culture work and additional metabolomics was it 55 possible to increase our understanding of TMAO bioconversions by the human gut microbiota. 56 2 bioRxiv preprint doi: https://doi.org/10.1101/225581; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 57 BACKGROUND 58 Dietary methylamines such as choline, trimethylamine N-oxide (TMAO), 59 phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish, 60 nuts and eggs. It has long been known that gut bacteria are able to use choline in a fermentation-like 61 process, with trimethylamine (TMA), ethanol, acetate and ATP among the known main end-products 62 [1–3]. TMA can be used by members of the order Methanomassiliicoccales (Archaea) present in the 63 human gut to produce methane [4], or taken up by the host. Microbially produced TMA derived from 64 PC is absorbed in the small intestine [5]. TMA diffuses into the bloodstream from the intestine via the 65 hepatic vein to hepatocytes, where it is converted to trimethylamine N-oxide (TMAO) by hepatic 66 flavin-containing mono-oxygenases (FMOs) [6]. The bulk of TMAO, and lesser amounts of TMA, 67 derived from dietary methylamines can be detected in urine within 6 h of ingestion, and both 68 compounds are excreted in urine but not faeces [7,8]. TMAO can also be detected in human skeletal 69 muscle within 6 h of an oral dose of TMAO [8]. 70 TMAO present in urine and plasma is considered a biomarker for non-alcoholic fatty liver 71 disease (NAFLD), insulin resistance and cardiovascular disease (CVD) [9–12]. Feeding TMAO to 72 high-fat-fed C57BL/6 mice exacerbates impaired glucose tolerance, though the effect on the gut 73 microbiota is unknown [13]. Low plasma PC and high urinary methylamines (including TMAO) were 74 observed in insulin-resistant mice on a high-fat diet, leading to the suggestion microbial methylamine 75 metabolism directly influences choline bioavailability; however, the microbiota of the mice was not 76 studied nor was labeled choline used to determine the metabolic fate of TMA/TMAO [9]. Choline 77 bioavailability is thought to contribute to hepatic steatosis in patients [11]. Spencer et al. [11] 78 manipulated dietary choline levels in 15 females for 2 weeks and monitored changes in the fecal 79 microbiota during the intervention. They found the level of choline supplementation affected 80 Gammaproteobacteria (inhibited by high levels of dietary choline, negatively correlated with changes 81 in liver fat/spleen fat ratios) and Erysipelotrichi (positively correlated with changes in liver fat/spleen 82 fat ratios), suggesting baseline levels of these taxa may predict the susceptibility of an individual to 83 liver disease. However, the study was under-powered and requires replication with a much larger 84 cohort to fully examine the link between choline bioavailability and liver disease. High levels of 3 bioRxiv preprint doi: https://doi.org/10.1101/225581; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 85 circulating TMAO are associated with CVD [12], potentially via a platelet-mediated mechanism [14]. 86 Circulating TMAO has been suggested to play a role in protection from hyperammonemia in mice, 87 acting as an osmoprotectant, and from glutamate neurotoxicity in mice and primary cultures of 88 neurons [15,16]. Recently, chronic exposure to TMAO was shown to attenuate diet-associated 89 impaired glucose tolerance in mice, and reduce endoplasmic reticulum stress and adipogenesis in 90 adipocytes [10]. The relevance of these findings to human health remains to be determined. There is a 91 basal level of TMA and TMAO detected in human urine even in the absence of dietary 92 supplementation [17], suggesting use of (microbial and/or host) cell-derived choline or PC in the 93 intestinal tract by the gut microbiota. 94 While it is well established that choline [2,11,18], PC [2,12] and carnitine [19–22] are used 95 by the human and rodent gut microbiotas to produce TMA, little is known about the reduction of 96 TMAO (predominantly from fish) to TMA (or other compounds) by members of these consortia. 97 TMAO is found at concentrations of 20–120 mg per 100 g fish fillet [23]. Ingestion of fish by humans 98 leads to increased urinary excretion of dimethylamine (DMA) and TMA, from 5.6 to 24.1 and from 99 0.2 to 1.6 µmol/24 h/kg of body weight, respectively [24]. Individuals with trimethylaminuria (fish 100 odour syndrome), in which TMA is not detoxified to TMAO by hepatic FMOs, have been shown to 101 reduce 40–60 % of an oral dose of TMAO to TMA [25].

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